Advanced SearchSearch Tips
A novel cold-active lipase from Psychrobacter sp. ArcL13: gene identification, expression in E. coli, refolding, and characterization
facebook(new window)  Pirnt(new window) E-mail(new window) Excel Download
 Title & Authors
A novel cold-active lipase from Psychrobacter sp. ArcL13: gene identification, expression in E. coli, refolding, and characterization
Koo, Bon-Hun; Moon, Byung-Hern; Shin, Jong-Suh; Yim, Joung-Han;
  PDF(new window)
Recently, Psychrobacter sp. ArcL13 strain showing the extracellular lipase activity was isolated from the Chuckchi Sea of the Arctic Ocean. However, due to the low expression levels of the enzyme in the natural strain, the production of recombinant lipase is crucial for various applications. Identification of the gene for the enzyme is prerequisite for the production of the recombinant protein. Therefore, in the present study, a novel lipase gene (ArcL13-Lip) was isolated from Psychrobacter sp. ArcL13 strain by gene prospecting using PCR, and its complete nucleotide sequence was determined. Sequence analysis showed that ArcL13-Lip has high amino acid sequence similarity to lipases from bacteria of some Psychrobacter genus (84-90%) despite low nucleotide sequence similarity. The lipase gene was cloned into the bacterial expression plasmid and expressed in E. coli. SDS-PAGE analysis of the cells showed that ArcL13-Lip was expressed as inclusion bodies with a molecular mass of about 35 kDa. Refolding was achieved by diluting the unfolded protein into refolding buffers containing various additives, and the highest refolding efficiency was seen in the glucose-containing buffer. Refolded ArcL13-Lip showed high hydrolytic activity toward p-nitrophenyl caprylate and p-nitrophenyl decanoate among different p-nitrophenyl esters. Recombinant ArcL13-Lip displayed maximal activity at and pH 8.0 with p-nitrophenyl caprylate as a substrate. Activity assays performed at various temperatures showed that ArcL13-Lip is a cold-active lipase with about 40% and 73% of enzymatic activity at and , respectively, compared to its maximal activity at .
Psychrobacter sp. ArcL13;cold-active lipase;gene prospecting;refolding;
 Cited by
Arakawa, T. and Timasheff, S.N. 1982. Stabilization of protein structure by sugars. Biochemistry 21, 6536-6544. crossref(new window)

Bakermans, C., Ayala-del-rio, H.L., Ponder, M.A., Vishnivetskaya, T., Gilichinsky, D., Thomashow, M.F., and Tiedje, J.M. 2006. Psychrobacter cryohalolentis sp. nov. and Psychrobacter arcticus sp. nov., isolated from Siberian permafrost. Int. J. Syst. Evol. Microbiol. 56, 1285-1291. crossref(new window)

Bell, P.J., Sunna, A., Gibbs, M.D., Curach, N.C., Nevalainen, H., and Bergquist, P.L. 2002. Prospecting for novel lipase genes using PCR. Microbiology 148, 2283-2291. crossref(new window)

Cardenas, F., de Castro, M.S., Sanchez-Montero, J.M., Sinisterra, J.V., Valmaseda, M., Elson, S.W., and Alvarez, E. 2001. Novel microbial lipases: catalytic activity in reactions in organic media. Enzyme Microb. Technol. 28, 145-154. crossref(new window)

Chen, R.P., Guo, L.Z., and Dang, H.Y. 2011. Gene cloning, expression and characterization of a cold-adapted lipase from a psychrophilic deep-sea bacterium Psychrobacter sp. C18. World J. Microbiol. Biotechnol. 27, 431-441. crossref(new window)

Feller, G. 2013. Psychrophilic enzymes: from folding to function and biotechnology. Scientifica 2013, 512840.

Finnegan, P.M., Brumbley, S.M., O'Shea, M.G., Nevalainen, H., and Bergquist, P.L. 2005. Diverse dextranase genes from Paenibacillus species. Arch. Microbiol. 183, 140-147. crossref(new window)

Gerday, C., Aittaleb, M., Bentahir, M., Chessa, J.P., Claverie, P., Collins, T., D'Amico, S., Dumont, J., Garsoux, G., Georlette, D., et al. 2000. Cold-adapted enzymes: from fundamentals to biotechnology. Trends Biotechnol. 18, 103-107. crossref(new window)

Hartl, F.U., Bracher, A., and Hayer-Hartl, M. 2011. Molecular chaperones in protein folding and proteostasis. Nature 475, 324-332. crossref(new window)

Hottiger, T., De Virgilio, C., Hall, M.N., Boller, T., and Wiemken, A. 1994. The role of trehalose synthesis for the acquisition of thermotolerance in yeast. II. Physiological concentrations of trehalose increase the thermal stability of proteins in vitro. Eur. J. Biochem. 219, 187-193. crossref(new window)

Jaeger, K.E., Dijkstra, B.W., and Reetz, M.T. 1999. Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications of lipases. Annu. Rev. Microbiol. 53, 315-351. crossref(new window)

Jaeger, K.E., Ransac, S., Dijkstra, B.W., Colson, C., Vanheuvel, M., and Misset, O. 1994. Bacterial lipases. FEMS Microbiol. Rev. 15, 29-63. crossref(new window)

Joseph, B., Ramteke, P.W., and Thomas, G. 2008. Cold active microbial lipases: Some hot issues and recent developments. Biotechnol. Adv. 26, 457-470. crossref(new window)

Kim, S., Wi, A.R., Park, H.J., Kim, D., Kim, H.W., Yim, J.H., and Han, S.J. 2015. Enhancing extracellular lipolytic enzyme production in an arctic bacterium, Psychrobacter sp. ArcL13, by using statistical optimization and fed-batch fermentation. Prep. Biochem. Biotechnol. 45, 348-364. crossref(new window)

Kulakova, L., Galkin, A., Nakayama, T., Nishino, T., and Esaki, N. 2004. Cold-active esterase from Psychrobacter sp. Ant300: gene cloning, characterization, and the effects of Gly ${\rightarrow}$ Pro substitution near the active site on its catalytic activity and stability. Biochim. Biophys. Acta. 1696, 59-65. crossref(new window)

Lee, J.C. and Timasheff, S.N. 1981. The stabilization of proteins by sucrose. J. Biol. Chem. 256, 7193-7201.

Novototskaya-Vlasova, K., Petrovskaya, L., Kryukova, E., Rivkina, E., Dolgikh, D., and Kirpichnikov, M. 2013a. Expression and chaperone-assisted refolding of a new cold-active lipase from Psychrobacter cryohalolentis $K5^T$. Protein Expr. Purif. 91, 96-103. crossref(new window)

Novototskaya-Vlasova, K.A., Petrovskaya, L.E., Rivkina, E.M., Dolgikh, D.A., and Kirpichnikov, M.P. 2013b. Characterization of a cold-active lipase from Psychrobacter cryohalolentis $K5^T$ and its deletion mutants. Biochemistry (Mosc) 78, 385-394. crossref(new window)

Novototskaya-Vlasova, K., Petrovskaya, L., Yakimov, S., and Gilichinsky, D. 2012. Cloning, purification, and characterization of a cold-adapted esterase produced by Psychrobacter cryohalolentis K5T from Siberian cryopeg. FEMS Microbiol. Ecol. 82, 367-375. crossref(new window)

Park, I.H., Kim, S.H., Lee, Y.S., Lee, S.C., Zhou, Y., Kim, C.M., Ahn, S.C., and Choi, Y.L. 2009. Gene cloning, purification, and characterization of a cold-adapted lipase produced by Acinetobacter baumannii BD5. J. Microbiol. Biotechnol. 19, 128-135. crossref(new window)

Salameh, M.A. and Wiegel, J. 2007. Purification and characterization of two highly thermophilic alkaline lipases from Thermosyntropha lipolytica. Appl. Environ. Microbiol. 73, 7725-7731. crossref(new window)

Sarkar, P., Yamasaki, S., Basak, S., Bera, A., and Bag, P.K. 2012. Purification and characterization of a new alkali-thermostable lipase from Staphylococcus aureus isolated from Arachis hypogaea rhizosphere. Process Biochem. 47, 858-866. crossref(new window)

Shandilya, H., Griffiths, K., Flynn, E.K., Astatke, M., Shih, P.J., Lee, J.E., Gerard, G.F., Gibbs, M.D., and Bergquist, P.L. 2004. Thermophilic bacterial DNA polymerases with reverse-transcriptase activity. Extremophiles 8, 243-251. crossref(new window)

Singer, M.A. and Lindquist, S. 1998. Multiple effects of trehalose on protein folding in vitro and in vivo. Mol. Cell 1, 639-648. crossref(new window)

Smalas, A.O., Leiros, H.K., Os, V., and Willassen, N.P. 2000. Cold adapted enzymes. Biotechnol. Annu. Rev. 6, 1-57. crossref(new window)

Sunna, A. and Bergquist, P.L. 2003. A gene encoding a novel extremely thermostable 1,4-beta-xylanase isolated directly from an environmental DNA sample. Extremophiles 7, 63-70.

Suzuki, T., Nakayama, T., Kurihara, T., Nishino, T., and Esaki, N. 2002. Primary structure and catalytic properties of a cold-active esterase from a psychrotroph, Acinetobacter sp. strain no. 6. isolated from Siberian soil. Biosci. Biotechnol. Biochem. 66, 1682-1690. crossref(new window)

Vallejo, L.F. and Rinas, U. 2004. Strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins. Microb. Cell Fact. 3, 11. crossref(new window)

Zhang, A.J., Gao, R.J., Diao, N.B., Xie, G.Q., Gao, G., and Cao, S.G. 2009. Cloning, expression and characterization of an organic solvent tolerant lipase from Pseudomonas fluorescens JCM5963. J. Mol. Catal. B Enzym. 56, 78-84. crossref(new window)

Zhang, J., Lin, S., and Zeng, R.Y. 2007. Cloning, expression, and characterization of a cold-adapted lipase gene from an Antarctic deep-sea psychrotrophic bacterium, Psychrobacter sp. 7195. J. Microbiol. Biotechnol. 17, 604-610.

Zhao, J.C., Zhao, Z.D., Wang, W., and Gao, X.M. 2005. Prokaryotic expression, refolding, and purification of fragment 450-650 of the spike protein of SARS-coronavirus. Protein Expr. Purif. 39, 169-174. crossref(new window)